Laboratoire de Biochimie, Ecole polytechnique, CNRS, Université Paris-Saclay, 91128 Palaiseau cedex, France.
Nucleic Acids Res. 2018 Nov 16;46(20):11061-11074. doi: 10.1093/nar/gky850.
In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.
在古菌翻译起始过程中，由 aIF1、aIF1A、三元复合物（TC，e/aIF2-GTP-Met-tRNAiMet）和与小核糖体亚基结合的 mRNA 组成的起始前复合物（PIC）负责起始密码子的选择。许多古菌 mRNA 含有 Shine-Dalgarno（SD）序列，允许 PIC 预先定位在起始密码子附近。然而，低温电子显微镜研究表明，局部扫描可以确定起始密码子与 tRNA 反密码子的碱基配对。在这里，我们使用荧光各向异性表明 aIF1 和 mRNA 与 Pyrococcus abyssi 30S 具有协同结合作用。30S:mRNA:aIF1 的稳定性强烈依赖于 SD 序列。此外，足迹实验表明，含有 aIF1 的 PIC 显示出动态构象，tRNA 未牢固地容纳在 P 位。aIF1 诱导 PIC 不稳定有利于校对错误起始复合物。aIF1 离开后，PIC 的稳定性增加，反映出起始 tRNA 与起始密码子完全碱基配对。总之，我们的数据支持这样一种观点，即在真核生物和古菌中控制起始密码子选择的一些主要事件发生在一个共同的结构和功能核心内。然而，涉及 30S:mRNA 结合的 loop 1 序列中的独特特征表明，e/aIF1 在两个结构域中的功能存在调整。
